Nuclear import of proteins that is mediated by classical nuclear localization sequences (NLSs) involves the formation of a complex containing the substrate and importin alpha/beta in the cytoplasm, followed by the stepwise movement of this complex through the nuclear pore complex (NPC) to the nuclear interior. The small GTPase Ran plays an important but poorly understood role in this process. The long term goal of this work is to understand the molecular basis for signal mediated import through the NPC, and how this is coordinated with other cellular processes. This project will involve a detailed analysis of a number of key issues related to NLS-mediated nuclear import. First, the functions of Ran in nuclear import will be analyzed by electron microscopy of staged in vitro import assays involving gold-coupled substrate, as well as by biochemical and functional analysis of the interactions of Ran during discrete transport steps. Second, the interactions of importin beta during movement of a transport complex through the NPC will be analyzed by in vitro binding studies with discrete nucleoporins, by investigation of transport arrests obtained with importin beta mutants, and by biochemical analysis of the binding partners of importin beta under different conditions of transport arrest. Third, the functions of specific nucleoporins in the nuclear import pathway will be examined in permeabilized cell assays with domain-specific inhibitory antibodies and with cells expressing dominant negative mutants of nucleoporins. Since nuclear transport is integrally involved in gene expression, this project has direct relevance to a number of human health issues including cancer, viral pathogenesis, immunity and aging.